Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-01-06 to 2004-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C (dated 2000-05-19)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S2A and S2B (CPMP/ICH/141/95; CPMP/ICH/174/95)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): T002251
- Physical state: Solid
- Appearance: Brown powder
Specific details on test material used for the study:
Batch No.: 00410155
Aggregate State at Room Temperature: Solid
Colour: Brown
Purity: 96%
Stability in Solvent: Not indicated by the Sponsor
Storage: At room temperature
Expiration Date: May 05, 2004

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 5-7 weeks (males); 7-9 weeks (females)
- Weight at study initiation: males mean value 29.7g (SD +/- 2.2g); females mean value 25.0g (SD +/- 1.7g)
- Assigned to test groups randomly: yes, and identified by cage number
- Fasting period before study: the animals were starved over night but received water ad libitum
- Housing: single, Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 25 - 72%
- Photoperiod (hrs dark / hrs light): 12h light (artificial) / 12h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals
- Concentration of test material in vehicle: adequate spaced dose levels
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 20 ml/kg body weight orally.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was formulated in PEG 400.
Duration of treatment / exposure:
24h and 48h (only highest dose level)
Frequency of treatment:
The animals received the test item, the vehicle or the positive control substance once.
Post exposure period:
Examination of animals of all dose groups for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Negative control
24h sampling time
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Test group
24h sampling time
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Test group
24h sampling time
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Test group
24h and 48h sampling time
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Positive control
24h sampling time
No. of animals per sex per dose:
Six males and six females were assigned to eaeh test group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: single oral administration
- Doses / concentrations: 40 mg/kg b.w. (volume administered 10 ml/kg b.w.)
- Other: dissolved in deionised water; solution prepared on day of administration

Examinations

Tissues and cell types examined:
bone marrow tissue - at least 2000 polychromatic erythrocytes were analysed per animal for micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24h after treatment. For the highest dose level an additional sample was taken at 48h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved over night but received water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item.
Sampling of the bone marrow was done 24h and 48h after treatment, respectively.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed with CO2 and cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resispended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide ws made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males / 5 females) per test group were evaluated as described. The remaining 6th animal of each group and sex is evaluated in case an animal of that group dies.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 males and 1 female died after treatment with 600 mg/kg b.w. in main experiment (no deaths at 600 mg/kg b.w. in pre-experiment)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 600, 750 and 1000 mg/kg b.w.
- Solubility: soluble
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, adbominal position, eyelid closure, ruffled fur, apathy, death
- Evidence of cytotoxicity in tissue analyzed: To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sampie and reported as the number of PCEs per 2000 erythrocytes.
- Harvest times: 24h and 48h (for highest dose level)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with T002251 were below or near to the value of the vehicle control group.
The following percentages of PCEs with micronuclei were calculated:
a) vehicle (24h): 0.055%
b) test item 150 mg/kg b.w. (24h): 0.075%
c) test item 300 mg/kg b.w. (24h): 0.070%
d) test item 600 mg/kg b.w. (24h): 0.060%
e) positive control 40 mg/kg b.w. (24h): 2.185%
f) test item 600 mg/kg b.w. (48h): 0.030%
- Ratio of PCE/NCE (for Micronucleus assay):
a) vehicle (24h): 1103/2000
b) test item 150 mg/kg b.w. (24h): 1042/2000
c) test item 300 mg/kg b.w. (24h): 1082/2000
d) test item 600 mg/kg b.w. (24h): 1088/2000
e) positive control 40 mg/kg b.w. (24h): 1123/2000
f) test item 600 mg/kg b.w. (48h): 863/2000
- Appropriateness of dose levels and route: determined via pre-experiment
- Statistical evaluation: no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.